Nf-kb inhibitors

ABSTRACT

The present invention provides novel compounds and methods for using them to treat diseases with aminothiophene inhibitors of IKK-β phosphorylation of IκB. In so doing these aminothiophene inhibitors block pathological activation of transcription factor NF-κB in which diseases excessive activation of NF-κB is implicated.

FIELD OF THE INVENTION

This invention relates in general to a method of inhibiting pathologicalactivation of the transcription factor NF-κB (nuclear factor-κB) usingaminothiophene compounds. Such methods are particularly useful fortreating diseases in which activation of NF-κB is implicated. Morespecifically, these methods may be used for inhibiting IKK-β (IκBkinase-β, also known as IKK-2) phosphorylation of IκB (inhibitoryprotein κB)—which prevents subsequent degradation and activation ofNF-κB dimers. Such methods are useful in the treatment of a variety ofdiseases associated with NF-κB activation including inflammatory andtissue repair disorders, particularly rheumatoid arthritis, inflammatorybowel disease, asthma and COPD (chronic obstructive pulmonary disease);osteoarthritis, osteoporosis and fibrotic diseases; dermatosis,including psoriasis, atopic dermatitis and ultraviolet radiation(UV)-induced skin damage; autoimmune diseases including systemic lupuseythematosus, multiple sclerosis, psoriatic arthritis, alkylosingspondylitis, tissue and organ rejection, Alzheimer's disease, stroke,atherosclerosis, restenosis, diabetes, glomerulonephritis, cancer,including Hodgkins disease, cachexia, inflammation associated withinfection and certain viral infections, including acquired immunedeficiency syndrome (AIDS), adult respiratory distress syndrome, andAtaxia Telangiestasia.

BACKGROUND OF THE INVENTION

Recent advances in scientific understanding of the mediators involved inacute and chronic inflammatory diseases and cancer have led to newstrategies in the search for effective therapeutics. Traditionalapproaches include direct target intervention such as the use ofspecific antibodies, receptor antagonists, or enzyme inhibitors. Recentbreakthroughs in the elucidation of regulatory mechanisms involved inthe transcription and translation of a variety of mediators have led toincreased interest in therapeutic approaches directed at the level ofgene transcription.

Nuclear factor κB (NF-κB) belongs to a family of closely related dimerictranscription factor complexes composed of various combinations of theRel/NF-κB family of polypeptides. The family consists of five individualgene products in mammals, RelA (p65), NF-κB1 (p50/p105), NF-κB1(p49/p100), c-Rel, and RelB, all of which can form hetero- orhomodimers. These proteins share a highly homologous 300 amino acid “Relhomology domain” which contains the DNA binding and dimerizationdomains. At the extreme C-terminus of the Rel homology domain is anuclear translocation sequence important in the transport of NF-κB fromthe cytoplasm to the nucleus. In addition, p65 and cRel possess potenttransactivation domains at their C-terminal ends.

The activity of NF-κB is regulated by its interaction with a member ofthe inhibitor IκB family of proteins. This interaction effectivelyblocks the nuclear localization sequence on the NF-κB proteins, thuspreventing migration of the dimer to the nucleus. A wide variety ofstimuli activate NF-κB through what are likely to be multiple signaltransduction pathways. Included are bacterial products (LPS), someviruses (HIV-1, HTLV-1), inflammatory cytokines (TNFα, IL-1),environmental and oxidative stress and DNA damaging agents. Apparentlycommon to all stimuli however, is the phosphorylation and subsequentdegradation of Iκb. IκB is phosphorylated on two N-terminal serines bythe recently identified IκB kinases (IKK-α and IKK-β). Site-directedmutagenesis studies indicate that these phosphorylations are criticalfor the subsequent activation of NF-κB in that once phosphorylated theprotein is flagged for degradation via the ubiquitin-proteasome pathway.Free from Iκb, the active NF-κB complexes are able to translocate to thenucleus where they bind in a selective manner to preferred gene-specificenhancer sequences. Included in the genes regulated by NF-κB are anumber of cytokines and chemokines, cell adhesion molecules, acute phaseproteins, immunoregualtory proteins, eicosanoid metabolizing enzymes andanti-apoptotic genes.

It is well-known that NF-κB plays a key role in the regulated expressionof a large number of pro-inflammatory mediators including cytokines suchas TNF, IL-1, IL-6 and IL-8, cell adhesion molecules, such as ICAM andVCAM, and inducible nitric oxide synthase (iNOS). Such mediators areknown to play a role in the recruitment of leukocytes at sites ofinflammation and in the case of iNOS, may lead to organ destruction insome inflammatory and autoimmune diseases.

The importance of NF-κB in inflammatory disorders is furtherstrengthened by studies of airway inflammation including asthma, inwhich NF-κB has been shown to be activated. This activation may underliethe increased cytokine production and leukocyte infiltrationcharacteristic of these disorders. In addition, inhaled steroids areknown to reduce airway hyperresponsiveness and suppress the inflammatoryresponse in asthmatic airways. In light of the recent findings withregard to glucocorticoid inhibition of NF-κB, one may speculate thatthese effects are mediated through an inhibition of NF-κB.

Further evidence for a role of NF-κB in inflammatory disorders comesfrom studies of rheumatoid synovium. Although NF-κB is normally presentas an inactive cytoplasmic complex, recent immunohistochemical studieshave indicated that NF-κB is present in the nuclei, and hence active, inthe cells comprising rheumatoid synovium. Furthermore, NF-κB has beenshown to be activated in human synovial cells in response to stimulationwith TNF-α or IL-1β. Such a distribution may be the underlying mechanismfor the increased cytokine and eicosanoid production characteristic ofthis tissue. See Roshak, A. K, et al., J. Biol. Chem., 271, 31496-31501(1996). Expression of IKK-β has been shown in synoviocytes of rheumatoidarthritis patients and gene transfer studies have demonstrated thecentral role of IKK-β in stimulated inflammatory mediator production inthese cells. See Aupperele et al. J. Immunology 1999. 163: 427-433 andAupperle et al. J. Immunology 2001; 166: 2705-11. More recently, theintra-articular administration of a wild type IKK-β adenoviral constructwas shown to cause paw swelling while intra-articular administration ofdominant-negative IKK-β inhibited adjuvant-induced arthritis in rat. SeeTak et al. Arthritis and Rheumatism 2001; 44: 1897-1907.

The NF-κB/Rel and IκB proteins are also likely to play a key role inneoplastic transformation and metastasis. Family members are associatedwith cell transformation in vitro and in vivo as a result ofoverexpression, gene amplification, gene rearrangements ortranslocations. In addition, rearrangement and/or amplification of thegenes encoding these proteins are seen in 20-25% of certain humanlymphoid tumors. Further, NF-κB is activated by oncogenic ras, the mostcommon defect in human tumors and blockade of NF-κB activation inhibitsras mediated cell transformation. In addition, a role for NF-κB in theregulation of apoptosis has been reported, strengthening the role ofthis transcription factor in the regulation of tumor cell proliferation.TNF, ionizing radiation and DNA damaging agents have all been shown toactivate NF-κB which in turn leads to the upregulated expression ofseveral anti-apoptotic proteins. Conversely, inhibition of NF-κB hasbeen shown to enhance apoptotic-killing by these agents in several tumorcell types. As this likely represents a major mechanism of tumor cellresistance to chemotherapy, inhibitors of NF-κB activation may be usefulchemotherapeutic agents as either single agents or adjunct therapy.Recent reports have implicated NF-κB as an inhibitor of skeletal celldifferentiation as well as a regulator of cytokine-induced musclewasting (Guttridge et al. Science; 2000; 289: 2363-2365.) furthersupporting the potential of NF-κB inhibitors as novel cancer therapies.

Several NF-κB inhibitors are described in C. Wahl, et al. J. Clin.Invest. 101(5), 1163-1174 (1998), R. W. Sullivan, et al. J. Med. Chem.41, 413-419 (1998), J. W. Pierce, et al. J. Biol. Chem. 272, 21096-21103(1997).

The marine natural product hymenialdisine is known to inhibit NF-κB.Roshak, A., et al., JPET, 283, 955-961 (1997). Breton, J. J andChabot-Fletcher, M. C., JPET, 282, 459-466 (1997).

Additionally, patent applications have been filed on aminothiopheneinhibitors of the IKK-2, see Callahan, et al., WO 2002030353; Baxter, etal., WO 2001058890, Faull, et al., WO 2003010158; Griffiths, et al.,WO2003010163; Fancelli, et al., WO 200198290; imidazole inhibitors ofIKK-2, see Callahan, et al., WO 200230423; anilinophenylpyrimidineinhibitors of IKK-2, see Kois, et al., WO 2002046171; β-carbolineinhbitors of IKK-2, see Ritzeler, et al., WO 2001068648, Ritzeler, etal., EP 1134221; Nielsch, et al. DE 19807993; Ritzeler, et al., EP1209158; indole inhbitors of IKK-2, see Ritzeler, et al., WO 2001030774;benzimidazole inhibitors of the IKK-2, see Ritzeler, et al., DE19928424; Ritzeler et al, WO 2001000610; aminopyridine inhibitors ofIKK-2, see Lowinger, et al, WO2002024679; Murata, et al, WO 2002024693;Murata, et al., WO2002044153; pyrazolaquinazoline inhibitors of IKK-2,see Beaulieu, et al., WO2002028860; Burke et al, WO2002060386, Burke, etal. U.S. 20030022898; quinoline inhibitors of IKK-2, Browner, et al.,WO2002041843, Browner, et al., U.S. 20020161004 andpyridylcyanoguanidine inhibitors of IKK-2, see Bjorkling, et al., WO2002094813, Binderup et al, WO 2002094322 and Madsen, et al., WO200294265. The natural products staurosporine, quercetin, K252a andK252b have been shown to be IKK-2 inhibitors, see Peet, G. W. and Li, J.J. Biol. Chem., 274, 32655-32661 (1999) and Wisniewski, D., et al.,Analytical Biochem. 274, 220-228 (1999). Synthetic inhibitors of IKK-2have also been described, see Burke, et al. J. Biol. Chem., 278,1450-1456 (2003) and Murata, et al., Bioorg. Med. Chem. Lett., 13,913-198 (2003) have described IKK-2 inhibitors.

U.S. Pat. No. 3,963,750 describes the preparation of certainaminothiophenes.

SUMMARY OF THE INVENTION

The present invention involves novel compounds and novel methods ofinhibiting the activation transcription factor NF-κB using the presentcompounds.

An object of the present invention is to provide a method for treatingdiseases which may be therapeutically modified by altering the activityof transcription factor NF-κB.

Accordingly, in the first aspect, this invention provides apharmaceutical composition comprising a compound according to Formula I.

In another aspect, this invention provides a method of treating diseasesin which the disease pathology may be therapeutically modified byinhibiting phosphorylation and subsequent degradation of IκB by IKK-β.

In still another aspect, this invention provides a method of treatingdiseases in which the disease pathology may be therapeutically modifiedby inhibiting pathological activation of NF-κB.

In a particular aspect, this invention provides methods for treating avariety of diseases associated with NF-κB activation includinginflammatory and tissue repair disorders, particularly rheumatoidarthritis, inflammatory bowel disease, asthma and COPD (chronicobstructive pulmonary disease) osteoarthritis, osteoporosis and fibroticdiseases, dermatosis, including psoriasis, atopic dermatitis andultraviolet radiation (UV)-induced skin damage; autoimmune diseasesincluding systemic lupus eythematosus, multiple sclerosis, psoriaticarthritis, alkylosing spondylitis, tissue and organ rejection,Alzheimer's disease, stroke, atherosclerosis, restenosis, diabetes,glomerulonephritis, cancer, including Hodgkins disease, cachexia,inflammation associated with infection and certain viral infections,including acquired immune deficiency syndrome (AIDS), adult respiratorydistress syndrome and Ataxia Telangiestasia.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention are selected from Formula (I)herein below:

wherein:

-   R₁ represents NR₄R₅;-   R₂ represents CONH₂ or SO₂NH₂;-   R₃ represents up to three substituents selected from the group    consisting of halogen, C₁₋₄alkyl, NH₂, CF₃, OCF₃, O-alkyl, S-alkyl,    CN, CHO, SO₂-alkyl, and NO₂;-   R₄ represents H, C₁₋₂ alkyl;-   R₅ represents C(=A)NH₆, COR₇, or R₆;-   A represents O, S, or N;-   R₆ represents H, or C₁₋₂ alkyl;-   R₇ represents C₁₋₂ alkyl;-   L represents a linker D-E-D such that-   D represents a bond or C₁₋₄ alkyl;-   E represents    CONH, NHCO, COO, N, O, S, or    and-   G and I independently represent H, C₁₋₂ alkyl; or a pharamceutically    acceptable salt thereof, provided that the compound of formula (I)    is not    2-[(aminocarbonyl)amino]-5-{[(4-chlorophenyl)methyl]oxyl-3-thiophenecarboxamide.

Preferably R₂ is CONH₂.

Preferably R₅ is C(=A)NHR₆.

Preferably R₆ is H.

Preferably A is O.

Preferably E is

CONH, or

More preferably E is

or

Preferred compounds useful in the present invention include:

-   5-[(E)-phenyl)-ethenyl]-2-ureido-thiophene-3-carboxylic acid amide;-   5-[(E)-2-(4-Fluoro-phenyl)-ethenyl]-2-ureido-thiophene-3-carboxylic    acid amide;-   5-[(E)-2-(4-Chloro-phenyl)-ethenyl]-2-ureido-thiophene-3-carboxylic    acid amide;-   5-Phenethyl-2-ureido-thiophene-3-carboxylic acid amide;-   5-Benzyl-2-ureido-thiophene-3-carboxylic acid amide;-   5-(1-Phenyl-ethyl)-2-ureido-thiophene-3-carboxylic acid amide;-   5-Phenylethynyl-2-ureido-thiophene-3-carboxylic acid amide;-   5-(4-Fluorophenylethynyl)-2-ureido-thiophene-3-carboxylic acid    amide;-   5-(4-Ethylphenylethynyl)₂-ureido-thiophene-3-carboxylic acid amide;-   5-(4-Methoxyphenylethynyl)-2-ureido-thiophene-3-carboxylic acid    amide;-   5-(4-Chlorophenylethynyl)-2-ureido-thiophene-3-carboxylic acid    amide;-   5-(4-Trifluoromethylphenylethynyl)-2-ureido-thiophene-3-carboxylic    acid amide;-   5-(3-Trifluoromethylphenylethynyl)-2-ureido-thiophene-3-carboxylic    acid amide; and-   5-Acetylamino-thiophene-2,4-dicarboxylic acid 4-amide    2-[(3-chloro-phenyl)-amide]; or a pharmaceutically acceptable salt    thereof.

This invention provides methods for treating a variety of diseasesassociated with NF-κB activation including inflammatory and tissuerepair disorders; particularly rheumatoid arthritis, inflammatory boweldisease, asthma and COPD (chronic obstructive pulmonary disease)osteoarthritis, osteoporosis and fibrotic diseases; dermatosis,including psoriasis, atopic dermatitis and ultraviolet radiation(UV)-induced skin damage; autoimmune diseases including systemic lupuseythematosus, multiple sclerosis, psoriatic arthritis, alkylosingspondylitis, tissue and organ rejection, Alzheimer's disease, stroke,atherosclerosis, restenosis, diabetes, glomerulonephritis, cancer,including Hodgkins disease, cachexia, inflammation associated withinfection and certain viral infections, including aquired immunedeficiency syndrome (AIDS), adult respiratory distress syndrome, andAtaxia Telangiestasia.

Definitions

The present invention includes all hydrates, solvates, complexes andprodrugs of the compounds of this invention. Prodrugs are any covalentlybonded compounds, which release the active parent, drug according toFormula I in vivo. If a chiral center or another form of an isomericcenter is present in a compound of the present invention, all forms ofsuch isomer or isomers, including enantiomers and diastereomers, areintended to be covered herein. Inventive compounds containing a chiralcenter may be used as a racemic mixture, an enantiomerically enrichedmixture, or the racemic mixture may be separated using well-knowntechniques and an individual enantiomer may be used alone. In cases inwhich compounds have unsaturated carbon-carbon double bonds, both thecis (Z) and trans (E) isomers are within the scope of this invention. Incases wherein compounds may exist in tautomeric forms, such as keto-enoltautomers, each tautomeric form is contemplated as being included withinthis invention whether existing in equilibrium or predominantly in oneform.

The meaning of any substituent at any one occurrence in Formula I or anysubformula thereof is independent of its meaning, or any othersubstituent's meaning, at any other occurrence, unless specifiedotherwise.

As used herein, “alkyl” refers to an optionally substituted hydrocarbongroup joined by single carbon-carbon bonds and having 1-6 carbon atomsjoined together. The alkyl hydrocarbon group may be linear, branched orcyclic, saturated or unsaturated. Substituents on optionally substitutedalkyl are selected from the group consisting of aryl, OH, O-alkyl, CO,halogen, CF₃, and OCF₃.

As used herein, “aryl” refers to an optionally substituted aromaticgroup with at least one ring having a conjugated pi-electron system,containing up to two conjugated or fused ring systems. Aryl includescarbocyclic aryl, and biaryl groups, all of which may be optionallysubstituted. Substituents are selected from the group consisting ofhalogen, C₁₋₄ alkyl, NH₂, OCF₃, CF₃, O-alkyl, S-alkyl, CN, CHO,SO₂-alkyl and NO₂.

As used herein, “heteroaryl” refers to an optionally substitutedaromatic group with at least one ring having a conjugated-pi-electronsystem, containing up to two conjugated or fused ring systems and 1-3heteroatoms selected from O, S and N. Heteroaryl includes carbocyclicheteroarylaryl, aryl-heteroaryl and biheteroarylaryl groups, all ofwhich may be optionally substituted. Preferred aryl include phenyl andnaphthyl. More preferred aryl include phenyl. Preferred substituents areselected from the group consisting of halogen, C₁₋₄ alkyl, NH₂, OCF₃,CF₃, O-alkyl, S-alkyl, CN, CHO, SO₂-alkyl and NO₂. Examples ofheteroaryl rings included pyrrole, furan, thiophene, indole, isoindole,benzofuran, isobenzofuran, benzothiphene, pyridine, quinoline,isoquinoline, quinolizine, pyrazole, imidazole, isoxazole, oxazole,isothiazole, thiazole, pyridazine, pyrimidine, and pyrazine.

As used herein “halogen” refers to include F, Cl, Br, and I.

Methods of Preparation

The following methods and examples are intended to be illustrative ofthe present invention but not limiting in any way.

The general preparation of compounds described in Formula 1 is shown inSchemes 1, 2, 3, and 4. The key 5-bromo- or 5-iodo-2-aminothiopheneintermediate was prepared from the corresponding protected2-aminothiophene by treatment with N-bromosuccinimide (NBS) oriodochloride (ICI), respectively (Scheme 1). Utilizing the 5-bromo- or5-iodo-2-aminothiophene, analogs with olefin linker were prepared viaSuzuki coupling; analogs with an acetylene linker at 5-position wereprepared via Sonogashira coupling; analogs with amide, ester, thioesterlinker at 5-position were prepared via palladium catalyzed carbonylation(Scheme 2). Alternately, 2-aminothiophene 3-carboxylic amides can beprepared by reacting 2-cyanoacetamide with [1,4]-dithiane-2,5-diolfollowed by the protection with trichloroacetyl isocyanate. Theprotected thiophene is then halogenated by N-bromosuccinimide oriodochloride. Deprotection in sodium carbonate aqueous solutionfurnished the 5-bromo or 5-iodo-2-ureidothiopehene-3-carboxylic amide(Scheme 3). Aminothiophene with alkyl linkers at 5-position can beprepared by reacting the required aldehydes with 2-cyano acetamidefollowed by the reaction with chlorosulfonyl isocynate to give the2-ureido-3-amidethiophenes with alkyl linker at 5-position (Scheme 4).

General Procedures

Nuclear magnetic resonance spectra were recorded at 400 MHz using on aBruker AC 400 spectrometer. CDCl₃ is deuteriochloroform, DMSO-d₆ ishexadeuteriodimethylsulfoxide, and CD₃OD is tetradeuteriomethanol.Chemical shifts are reported in parts per million (d) downfield from theinternal standard tetramethylsilane. Abbreviations for NMR data are asfollows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,dd=doublet of doublets, dt=doublet of triplets, app=apparent, br=broad.J indicates the NMR coupling constant measured in Hertz. Mass spectrawere collected on a LCMS system using a Sciex API 150EX instrument [1×40mm Aquasil (C18) column, gradient 4.5%-90% acetonitdile-water (0.02%TFA) over 3.2 min, detection by mass, UV at 214 nm and by evaporativelight-scattering. Mass was determined using electrospray ionizationtechniques. All temperatures are reported in degree Celsius. AnaltechSilica Gel GF and E. Merck Silica Gel 60 F-254 thin layer plates wereused for thin layer chromatography. Both flash and gravitychromatography were carried out on E. Merck Kieselgel 60 (230-400 mesh)silica gel. Preparative HPLC were performed using a Gilson PreparativeSystem using a 50×20 mml S-5 μC18 reverse phase column eluting with a10-80 gradient (0.1% TFA in acetonitrile/0.1% aqueous TFA).

The following examples are intended to be illustrative of the presentinvention but not limiting in any way.

EXAMPLE 1 Preparation 5-Bromo-2-Ureido-thiophene-3-carboxylic Acid Amidea). 2-Amino-thiophene-3-carboxylic Acid Amide

Triethylamine (36 mL, 256 mmol) was added to the solution of2-cyanoacetamide (10.8 g, 128 mmol) and [1,4]-dithiane-2,5-diol (19.5 g,128 mmol) in ethanol (400 mL). The resulting reaction mixture wasstirred at room temperature for 5 min, then heated at reflux for 2 h.After the reaction mixture is cooled to room temperature, it wasfiltered and concentrated under vacuum. The residue was then taken upinto ethyl acetate (400 mL) and washed with aqueous sodium hydroxide (1M, 1×200 mL), water (2×200 mL) and brine (1×200 mL). The organic layerwas dried over anhydrous sodium sulfate and concentrated under vacuum togive the above titled compound as a light yellow solid (16 g, 113 mmol,88% yield). LC-MS [M+H]⁺ m/z 143.

b) 2-[3-(2,2,2-Trichloro-ethanoyl)-ureido]-thiophene-3-carboxylic AcidAmide

Trichloroacetyl isocyanate (10 g, 53 mmol) was added dropwise to thesolution of 2-amino-thiophene-3-carboxylic acid amide (3.8 g, 26.5 mmol)in pyridine (110 mL) at 0° C. The resulting reaction mixture was stirredat 0° C. The temperature of the reaction mixture was raised to roomtemperature over a 3 h period until LC-MS showed no remaining startingmaterial. Diethyl ether (100 mL) was added to the reaction mixture andthe resulting precipitate was filtered and dried under vacuum to givethe above titled compound as a light grey solid (8.7 g, 26.3 mmol, 99%yield).

c)5-Bromo-2-[3-(2,2,2-trichloro-ethanoyl)-ureido]-thiophene-3-carboxylicAcid Amide

N-Bromosuccinimide (1.56 g, 8.8 mmol) was added to a solution of2-[3-(2,2,2-trichloro-ethanoyl)-ureido]-thiophene-3-carboxylic acidamide (2.9 g, 8.8 mmol) in acetic acid (30 mL) and chloroform (10 mL) atroom temperature. The resulting solution was stirred under nitrogen withan aliquot was taken for LC-MS every 10 min. After 30 min., the LC-MSshowed no remaining starting material. Diethyl ether (80 mL) was addedto the solution and the resulting precipitate was filtered and driedunder vacuum to give the above titled compound as a light brown solid(3.5 g, 8.55 mmol, 97% yield).

d) 5-Bromo-2-ureido-thiophene-3-carboxylic Acid Amide

Sodium carbonate solution (10% aqueous, 30 ml) was added to a solutionof5-bromo-2-[3-(2,2,2-trichloro-ethanoyl)-ureido]-thiophene-3-carboxylicacid amide (1.0 g, 2.44 mmol) in EtOH (10 mL). The resulting reactionmixture was stirred under nitrogen overnight. After one third of thesolvent was removed under vacuum, the reaction mixture was filtered. Theprecipitate was washed with water (3×50 mL) and dried in vacuum to givethe above titled compound as a light brown solid (510 mg, 2.16 mmol, 89%yield).

EXAMPLE 2 Preparation of5-[(E)-Phenyl)-ethenyl]-2-ureido-thiophene-3-carboxylic Acid Amide 5

A solution of 5-bromo-2-ureido-thiophene-3-carboxylic acid amide (0.066g, 0.25 mmol) in absolute ethanol (2 mL) and toluene (2 mL) in a 5 mLreaction vial was treated with potassium acetate (0.05 g, 0.5 mmol),tetrakis (triphenylphosphine) palladium (0.015 g, 5 mole %), andtrans-2-phenylethenylboronic acid (0.041 g, 0.28 mmol). The reactionvial was then sealed and heat at 90-95° C. for 12 h. The solvent wasremoved under vacuum and the residue was taken up into DMSO (5 mL),filtered and purified utilizing a Gilson preparative HPLC system to givethe above titled product as a light brown solid (29 mg, 0.1 mmol, 40%yield). LC-MS [M+H]⁺ m/z 288.

EXAMPLE 3 Preparation of5-[(E)-2-(4-Fluoro-phenyl)-ethenyl]-2-ureido-thiophene-3-carboxylic AcidAmide

5-[(E)-2-(4-Fluoro-phenyl)-ethenyl]-2-ureido-thiophene-3-carboxylic acidamide was prepared by the same procedure as described in Example 2except that trans-2-phenylethenylboronic acid was replaced withtrans-2(4-fluorophenyl)vinylboronic acid. The above titled compound wasisolated as a light brown solid (31 mg, 0.1 mmol, 40% yield). ESMS[M+H]⁺ m/z 306.

EXAMPLE 4 Preparation of5-[(E)-2-(4-Chloro-phenyl)-ethenyl]-2-ureido-thiophene-3-carboxylic AcidAmide

5-[(E)-2-(4-Cloro-phenyl)-ethenyl]-2-ureido-thiophene-3-carboxylic acidamide was prepared using the same procedure as described in Example 2except that trans-2-phenylethenylboronic acid was replaced withtrans-2-(4-chlorophenyl)ethenylboronic acid. The above titled compoundwas isolated as a light brown solid (28 mg, 0.087 mmol, 35% yield). ESMS[M+H]⁺ m/z 322.

EXAMPLE 5 Preparation of 5-Phenethyl-2-ureido-thiophene-3-carboxylicAcid Amide

Palladium on carbon (10%, 0.01 g) was added to a solution of5-[(E)-styryl)-vinyl]-2-ureido-thiophene-3-carboxylic acid amide (0.02g, 0.06 mmol) in acetic acid (1 mL) and ethanol (1 mL) and the resultingreaction mixture was placed into a Parr reactor and treated with 20 psiH₂ for 72 h. The reaction mixture was then filtered through Celite andpurified by flash chromatography (silica gel, 100% ethyl acetate) togive the above titled compound as a white solid (5 mg, 25% yield). ESMS[M+H]⁺ m/z 290.

EXAMPLE 6 Preparation of 5-Benzyl-2-ureido-thiophene-3-carboxylic AcidAmide a) 2-Amino-5-benzyl-thiophene-3-carboxylic Acid AmideTrifluoroacetate

Triethyl amine (0.28 mL, 2.0 mmol) was added dropwise to a mixture of2-cyano-acetamide (0.168 g, 2.0 mmol), sulfur (0.064 g, 2.0 mmol) and3-phenyl propionaldehyde (0.29 g, 2.0 mmol) in DMF (8 mL) and resultingreaction mixture was stirred at room temperature overnight. The reactionmixture was then filtered and purified utilizing a Gilson preparativeHPLC to give the above titled compound as light yellow solid (0.25 g,1.06 mmol, 53% yield). LC-MS [M+H]+m/z 233.

b) 5-Benzyl-2-ureido-thiophene-3-carboxylic Acid Amide

Chlorosulfonyl isocyanate (0.09 mL, 1.03 mmol) was added dropwise undernitrogen to a solution of 2-amino-5-benzyl-thiophene-3-carboxylic acidamide (0.2 g, 0.86 mmol) in dry dichloromethane (9 mL) at 0° C. and. Thetemperature of the resulting stirred reaction mixture was slowly raisedfrom 0° C. to room temperature over 1 h. Once the LC-MS showed nostarting material remaining, the reaction was quenched with water (1 mL)and stirred for 10 additional min. The solvent was removed under vacuumand the residue was taken up into DMSO (2 mL) and purified utilizing aGilson preparative HPLC to give the above titled compound as a lightbrown solid (0.16 g, 67%). LC-MS [M+H]⁺ m/z 276.

EXAMPLE 7 Preparation of5-(1-Phenyl-ethyl)-2-ureido-thiophene-3-carboxylic Acid Amide a)2-Amino-5-(1-phenyl-ethyl)-thiophene-3-carboxylic Acid AmideTrifluoroacetate

2-Amino-5-(1-phenyl-ethyl)-thiophene-3-carboxylic acid amidetrifluoroacetate was prepared by the same procedure as described inExample 6a except that 3-phenylpropionaldehyde was replaced with3-phenylbutyraldehyde to give the above titled compound as a light brownsolid (17% yield). LC-MS [M+H]⁺ m/z 247.

b) 5-(1-Phenyl-ethyl)-2-ureido-thiophene-3-carboxylic Acid Amide

5-(1-Phenyl-ethyl)-2-ureido-thiophene-3-carboxylic acid amide wasprepared by the same procedure as Example 6b except that2-amino-5-benzyl-thiophene-3-carboxylic acid amide was replaced with2-amino-5-(1-phenyl-ethyl)-thiophene-3-carboxylic acid amide to give theabove titled compound as a light brown solid (87% yield). LC-MS [M+H]⁺m/z 290.

EXAMPLE 8 Preparation of 5-Phenylethynyl-2-ureido-thiophene-3-carboxylicAcid Amide

5-Bromo-2-ureido-thiophene-3-carboxylic acid amide (0.264 g, 1.0 mmol),copper (I) iodide (0.005 g), triphenyl phosphene (0.030 g, 15 mole %),palladium acetate (0.010 g, 5% mole %), triethyl amine (1 mL) andphenylacetylene (0.1 g, 1.1 mmol) in DMF (4 mL) was added to a 9 mLreaction vial. The reaction vial was sealed and heated to 100° C. in aSmith Microwave Synthesizer for 20 min. Water (5 mL) and ethyl acetate(20 mL) were added to the reaction mixture, the organic phase wasseparated and then washed with brine (2×10 mL) and water (1×10 mL), anddried over anhydrous magneium sulfate. After filtration, the solvent wasremoved under vacuum and the crude residue was taken into DMSO (5 mL)and purified utilizing a Gilson preparative HPLC to give the abovetitled compound as a light brown solid (0.050 g, 0.18 mmol, 18% yield).LC-MS [M+H]⁺ m/z 286.

EXAMPLE 9 Preparation of5-(4-Fluorophenylethynyl)-2-ureido-thiophene-3-carboxylic Acid Amide

5-(4-Fluorophenylethynyl)-2-ureido-thiophene-3-carboxylic acid amide wasprepared by the same procedure as Example 8 except phenylacetylene wasreplaced with 4-fluorophenylacetylene to give the above titled compoundas a light brown solid. LC-MS [M+H]⁺ m/z 304.

EXAMPLE 10 Preparation of5-(4-Ethylphenylethynyl)-2-ureido-thiophene-3-carboxylic Acid Amide

5-(4-Ethylphenylethynyl)-2-ureido-thiophene-3-carboxylic acid amide wasprepared by the same procedure as Example 8 except phenylacetylene wasreplaced with 4-ethylphenylacetylene to give the above titled compoundas a light brown solid. LC-MS [M+H]⁺ m/z 314.

EXAMPLE 11 Preparation of5-(4-Methoxyphenylethynyl)-2-ureido-thiophene-3-carboxylic Acid Amide

5-(4-Methoxyphenylethynyl)-2-ureido-thiophene-3-carboxylic acid amidewas prepared by the same procedure as Example 8 except phenylacetylenewas replaced with 4-methoxyphenylacetylene to give the above titledcompound as a light brown solid. LC-MS [M+H]⁺ m/z 316.

EXAMPLE 12 Preparation of5-(4-Chlorophenylethynyl)-2-ureido-thiophene-3-carboxylic Acid Amide

5-(4-Chlorophenylethynyl)-2-ureido-thiophene-3-carboxylic acid amide wasprepared by the same procedure as Example 8 except phenylacetylene wasreplaced with 4-chlorophenylacetylene to give the above titled compoundas a light brown solid. LC-MS [M+H]⁺ m/z 320.

EXAMPLE 13 Preparation of5-(4-Trifluoromethylphenylethynyl)-2-ureido-thiophene-3-carboxylic AcidAmide

5-(4-Trifluoromethylphenylethynyl)-2-ureido-thiophene-3-carboxylic acidwas prepared by the same procedure as Example 8 except phenylacetylenewas replaced with 4-trifluoromethylphenylacetylene to give the abovetitled compound as a light brown solid. LC-MS [M+H]⁺ m/z 354.

EXAMPLE 14 Preparation of5-(3-Trifluoromethylphenylethynyl)-2-ureido-thiophene-3-carboxylic AcidAmide

5-(3-Trifluoromethylphenylethynyl)₂-ureido-thiophene-3-carboxylic acidamide was prepared by the same procedure as Example 8 exceptphenylacetylene was replaced with 3-trifluoromethylphenylacetylene togive the above titled compound as a light brown solid. LC-MS [M+H]⁺ m/z354.

EXAMPLE 15 Preparation of 2-Acetylamino-5-bromo-thiophene-3-carboxylicAcid Amide a). 2-Acetylamino-Thiophene-3-carboxylic Acid Amide

Acetyl chloride (4.2 mL, 59 mmol) was added dropwise to a solution of2-amino-thiophene-3-carboxylic acid amide (7.7 g, 54.2 mmol) in pyridine(100 mL) at 0° C. The temperature of the reaction mixture was raised toroom temperature over a 3 h period until no remaining starting materialwas present based on LC-MS. The solvent was then removed under vacuum togive the above titled compound as brown solid (9.7 g, 52.7 mmol, 97.2%yield). LCMS [M+H]]⁺ m/z 185.

b). 2-Acetylamino-5-bromo-thiophene-3-carboxylic Acid Amide

2-Acetylamino-thiophene-3-carboxylic acid amide (3.89 g, 27.4 mmol) wassuspended in the aqueous solution of sodium acetate (2.47 g, 30.1mmol)(136 mL). Bromine (1.4 mL, 27.4 mmol) was added dropwise to thesolution. The resulting solution was stirred under nitrogen overnight.Once the LC-MS showed no remaining starting materials, the reactionmixture was then filtered and washed with water (50 mL). The filtratewas then purified utilizing a Gilson preparative. HPLC to give the abovetitled compound as a brown solid (1 g, 13.8%). LC-MS [M+H]⁺ m/z 263.

EXAMPLE 16 Preparation of 5-Acetylamino-thiophene-2,4-dicarboxylic acid4-amide-2-[(3-chloro-phenyl)-amide]

2-Acetylamino-5-bromo-thiophene-3-carboxylic acid amide (0.1 g, 0.38mmol), bis-triphenyl phosphene palladium(II) chloride (0.013 g, 5 mole%), diisopropylethylamine (0.16 g, 1.2 mmol), water (0.015 mL, 0.87mmol), 3-chloroaniline (0.12 g, 0.78 mmol) in NMP (4 mL) was added to a9 mL reaction vial. Carbon monoxide was bubbled through the reactionmixture via a plastic balloon filled with carbon monoxide gas. Thereaction vial was sealed and stirred at 90° C. for 12 h. Water (5 mL)and ethyl acetate (20 mL) were added to the reaction mixture, theorganic phase was separated and then washed with brine (2×10 mL) andwater (1×10 mL), and dried over anhydrous magnesium sulfate. Afterfiltration, the solvent was removed under vacuum and the crude residuewas taken into DMSO (5 mL) and purified utilizing a Gilson preparativeHPLC to give the above titled product as a light brown solid (0.020 g,0.17 mmol, 16% yield). LC-MS [M+H]⁺ m/z 338.

This invention provides a pharmaceutical composition, which comprises acompound according to Formula I and a pharmaceutically acceptablecarrier, diluent or excipient. Accordingly, the compounds of Formula Imay be used in the manufacture of a medicament. Pharmaceuticalcompositions of the compounds of Formula I prepared as hereinbeforedescribed may be formulated as solutions or lyophilized powders forparenteral administration. Powders may be reconstituted by addition of asuitable diluent or other pharmaceutically acceptable carrier prior touse. The liquid formulation may be a buffered, isotonic, aqueoussolution. Examples of suitable diluents are normal isotonic salinesolution, standard 5% dextrose in water or buffered sodium or ammoniumacetate solution. Such formulation is especially suitable for parenteraladministration, but may also be used for oral administration orcontained in a metered dose inhaler or nebulizer for insufflation. Itmay be desirable to add excipients such as polyvinylpyrrolidone,gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol,sodium chloride or sodium citrate.

Alternately, these compounds may be encapsulated, tableted or preparedin an emulsion or syrup for oral administration. Pharmaceuticallyacceptable solid or liquid carriers may be added to enhance or stabilizethe composition, or to facilitate preparation of the composition. Solidcarriers include starch, lactose, calcium sulfate dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. Liquid carriers include syrup, peanut oil, olive oil, salineand water. The carrier may also include a sustained release materialsuch as glyceryl monostearate or glyceryl distearate, alone or with awax. The amount of solid carrier varies but, preferably, will be betweenabout 20 mg to about 1 g per dosage unit. The pharmaceuticalpreparations are made following the conventional techniques of pharmacyinvolving milling, mixing, granulating, and compressing, when necessary,for tablet forms; or milling, mixing and filing for hard gelatin capsuleforms. When a liquid carrier is used, the preparation will be in theform of a syrup, elixir, emulsion or an aqueous or non-aqueoussuspension. Such a liquid formulation may be administered directly p.o.or filled into a soft gelatin capsule.

Typical compositions for inhalation are in the form of a dry powder,solution, suspension or emulsion. Administration may for example be bydry powder inhaler (such as unit dose or multi-dose inhaler, e.g., asdescribed in U.S. Pat. No. 5,590,645 or by nebulisation or in the formof a pressurized aerosol. Dry powder compositions typically employ acarrier such as lactose, trehalose or starch. Compositions fornebulisation typically employ water as vehicle. Pressurized aerosolstypically employ a propellant such as dichlorodifluoromethane,trichlorofluoromethane or, more preferably, 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane or mixtures thereof. Pressurizedaerosol formulations may be in the form of a solution (perhaps employinga solubilising agent such as ethanol) or a suspension which may beexcipient free or employ excipients including surfactants and/orco-solvents (e.g. ethanol). In dry powder compositions and suspensionaerosol compositions the active ingredient will preferably be of a sizesuitable for inhalation (typically having mass median diameter (MMD)less than 20 microns, e.g., 1-10 especially 1-5 microns). Size reductionof the active ingredient may be necessary, e.g., by micronisation.

Pressurized aerosol compositions will generally be filled into canistersfitted with a valve, especially a metering valve. Canisters mayoptionally be coated with a plastics material e.g. a fluorocarbonpolymer as described in WO96/32150. Canisters will be fitted into anactuator adapted for buccal delivery.

Typical compositions for nasal delivery include those mentioned abovefor inhalation and further include non-pressurized compositions in theform of a solution or suspension in an inert vehicle such as wateroptionally in combination with conventional excipients such as buffers,anti-microbials, tonicity modifying agents and viscosity modifyingagents which may be administered by nasal pump.

For rectal administration, the compounds of this invention may also becombined with excipients such as cocoa butter, glycerin, gelatin orpolyethylene glycols and molded into a suppository.

The methods of the present invention include topical inhaled andintracolonic administration of the compounds of Formula I. By topicaladministration is meant non-systemic administration, including theapplication of a compound of the invention externally to the epidermis,to the buccal cavity and instillation of such a compound into the ear,eye and nose, wherein the compound does not significantly enter theblood stream. By systemic administration is meant oral, intravenous,intraperitoneal and intramuscular administration. The amount of acompound of the invention (hereinafter referred to as the activeingredient) required for therapeutic or prophylactic effect upon topicaladministration will, of course, vary with the compound chosen, thenature and severity of the condition being treated and the animalundergoing treatment, and is ultimately at the discretion of thephysician.

While it is possible for an active ingredient to be administered aloneas the raw chemical, it is preferable to present it as a pharmaceuticalformulation. The active ingredient may comprise, for topicaladministration, from 0.01 to 5.0 wt % of the formulation.

The topical formulations of the present invention, both for veterinaryand for human medical use, comprise an active ingredient together withone or more acceptable carriers therefor and optionally any othertherapeutic ingredients. The carrier must be “acceptable” in the senseof being compatible with the other ingredients of the formulation andnot deleterious to the recipient thereof.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required such as: liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to a suitable container,which is then sealed and sterilized by autoclaving, or maintaining at90-100° C. for half an hour. Alternatively, the solution may besterilized by filtration and transferred to the container by an aseptictechnique. Examples of bactericidal and fungicidal agents suitable forinclusion in the drops are phenylmercuric nitrate or acetate (0.002%),benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient in finelydivided or powdered form, alone or in solution or suspension in anaqueous or non-aqueous fluid, with the aid of suitable machinery, with agreasy or non-greasy basis. The basis may comprise hydrocarbons such ashard, soft or liquid paraffin, glycerol, beeswax, a metallic soap, amucilage, an oil of natural origin such as almond, corn, arachis, castoror olive oil, wool fat or its derivatives, or a fatty acid such asstearic or oleic acid together with an alcohol such as propylene glycolor macrogols. The formulation may incorporate any suitable surfaceactive agent such as an anionic, cationic or non-ionic surface activeagent such as sorbitan esters or polyoxyethylene derivatives thereof.Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredientssuch as lanolin, may also be included.

Utility of the Present Invention

The compounds of Formula I are useful as inhibitors of the IKK-betakinase phosphorylation of IκB and as such are inhibitors of NF-κBactivation. The present method utilizes compositions and formulations ofsaid compounds, including pharmaceutical compositions and formulationsof said compounds.

The present invention particularly provides methods of treatment ofdiseases associated with inappropriate NF-κB activation, which methodscomprise administering to an animal, particularly a mammal, mostparticularly a human in need thereof one or more compounds of Formula I.The present invention particularly provides methods for treatinginflammatory and tissue repair disorders, particularly rheumatoidarthritis, inflammatory bowel disease, asthma and COPD (chronicobstructive pulmonary disease); osteoarthritis, osteoporosis andfibrotic diseases; dermatosis, including psoriasis, atopic dermatitisand ultraviolet radiation (UV)-induced skin damage, autoimmune diseasesincluding systemic lupus eythematosus, multiple sclerosis, psoriaticarthritis, alkylosing spondylitis, tissue and organ rejection,Alzheimer's disease, stroke, atherosclerosis, restenosis, diabetes,glomerulonephritis, cancer, including Hodgkins disease, cachexia,inflammation associated with infection and certain viral infections,including aquired immune deficiency syndrome (AIDS), adult respiratorydistress syndrome and Ataxia Telangiestasia.

For acute therapy, parenteral administration of one or more compounds ofFormula I is useful. An intravenous infusion of the compound in 5%dextrose in water or normal saline, or a similar formulation withsuitable excipients, is most effective, although an intramuscular bolusinjection is also useful. Typically, the parenteral dose will be about0.01 to about 50 mg/kg; preferably between 0.1 and 20 mg/kg, in a mannerto maintain the concentration of drug in the plasma at a concentrationeffective to inhibit IKK-beta and therefore activation of NF-κB. Thecompounds are administered one to four times daily at a level to achievea total daily dose of about 0.4 to about 80 mg/kg/day. The preciseamount of a compound used in the present method which is therapeuticallyeffective, and the route by which such compound is best administered, isreadily determined by one of ordinary skill in the art by comparing theblood level of the agent to the concentration required to have atherapeutic effect.

The compounds of Formula I may also be administered orally to thepatient, in a manner such that the concentration of drug is sufficientto inhibit IKK-beta and therefore activation of NF-κB or to achieve anyother therapeutic indication as disclosed herein. Typically, apharmaceutical composition containing the compound is administered at anoral dose of between about 0.1 to about 50 mg/kg in a manner consistentwith the condition of the patient. Preferably the oral dose would beabout 0.5 to about 20 mg/kg.

The compounds of Formula I may also be administered topically to thepatient, in a manner such that the concentration of drug is sufficientto inhibit IKK-beta and therefore activation of NF-κB or to achieve anyother therapeutic indication as disclosed herein. Typically, apharmaceutical composition containing the compound is administered in atopical formulation of between about 0.01% to about 5% w/w.

No unacceptable toxicological effects are expected when compounds of thepresent invention are administered in accordance with the presentinvention.

The ability of the compounds described herein to inhibit the activationof NF-κB is clearly evidenced in their ability to inhibit thephosphorylation of the N-terminal fragment of IκB-α by IKK-β. Thesecompounds also block the degradation of IκB-α and the nucleartranslocation of NF-κB in human monocyctes and other mammalian cellsupon activation of the cells with a pro-inflammatory stimuii (e.g.,TNF-α, LPS, etc.). In addition these compounds inhibit pro-inflammatorymediator production from LPS-stimulated human monocytes and stimulatedhuman primary synovial fibroblasts. The utility of the present NF-κBinhibitors in the therapy of diseases is premised on the importance ofNF-κB activation in a variety of diseases.

NF-κB plays a key role in the regulated expression of a large number ofpro-inflammatory mediators-including cytokines such as TNF, IL-1β, IL-6and IL-8 (Mukaida et al., 1990; Liberman and Baltimore, 1990; Matsusakaet al., 1993), cell adhesion molecules, such as ICAM and VCAM (Marui etal., 1993; Kawai et al., 1995; Ledebur and Parks, 1995), and induciblenitric oxide synthase (iNOS) (Xie et al., 1994; Adcock et al., 1994).Such mediators are known to play a role in the recruitment of leukocytesat sites of inflammation and in the case of iNOS, may lead to organdestruction in some inflammatory and autoimmune diseases(McCartney-Francis et al., 1993; Kleemann et al., 1993).

Evidence for an important role of NF-κB in inflammatory disorders isobtained in studies of asthmatic patients. Bronchial biopsies taken frommild atopic asthmatics show significant increases in the number of cellsin the submucosa staining for activated NF-κB, total NF-κB, andNF-κB-regulated cytokines such as GM-CSF and TNFα compared to biopsiesfrom normal non-atopic controls (Wilson et al., 1998). Furthermore, thepercentage of vessels expressing NF-κB immunoreactivity is increased asis IL-8 immunoreactivity in the epithelium of the biopsy specimens(Wilson et al., 1998). As such, inhibition of IL-8 production throughthe inhibition of NF-κB, as has been demonstrated by these compoundswould be predicted be beneficial in airway inflammation.

Recent studies suggest that NF-κB may also play a critical role in thepathogenesis of inflammatory bowel disease (IBD). Activated NF-κB isseen in colonic biopsy specimens from Chron's disease and ulcerativecolitis patients (Ardite et al., 1998; Rogler et al., 1998; Schreiber etal., 1998). Activation is evident in the inflamed mucosa but not inuninflamed mucosa (Ardite et al., 1998; Rogler et al., 1998) and isassociated with increased IL-8 mRNA expression in the same sites (Arditeet al., 1998). Furthermore, corticosteroid treatment strongly inhibitsintestinal NF-κB activation and reduces colonic inflammation (Ardite etal., 1998; Schreiber et al., 1998). Again, inhibition of IL-8 productionthrough the inhibition of NF-κB, as has been demonstrated by thesecompounds would be predicted be beneficial in inflammatory boweldisease.

Animal models of gastrointestinal inflammation provide further supportfor NF-κB as a key regulator of colonic inflammation. Increased NF-κBactivity is observed in the lamina propria macrophages in2,4,6,-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice withp65 being a major component of the activated complexes (Neurath et al.,1996; Neurath and Pettersson, 1997). Local administration of p65antisense abrogates the signs of established colitis in the treatedanimals with no signs of toxicity (Neurath et al., 1996; Neurath andPettersson, 1997). As such, one would predict that small moleculeinhibitors of NF-κB would be useful in the treatment of IBD.

Further evidence for a role of NF-κB in inflammatory disorders comesfrom studies of rheumatoid synovium. Although NF-κB is normally presentas an inactive cytoplasmic complex, recent immunohistochemical studieshave indicated that NF-κB is present in the nuclei, and hence active, inthe cells comprising human rheumatoid synovium (Handel et al., 1995;Marok et al., 1996; Sioud et al., 1998) and in animal models of thedisease (Tsao et al., 1997). The staining is associated with type Asynoviocytes and vascular endothelium (Marok et al., 1996). Furthermore,constitutive activation of NF-κB is seen in cultured synoviocytes(Roshak et al., 1996; Miyazawa et al., 1998) and in synovial cellcultures stimulated with IL-1β or TNFα (Roshak et al., 1996; Fujisawa etal., 1996; Roshak et al., 1997). Thus, the activation of NF-κB mayunderlie the increased cytokine production and leukocyte infiltrationcharacteristic of inflamed synovium. The ability of these compounds toinhibit NF-κB and thereby inhibit the production of pro-inflammatorymediators (e.g., cytokines and prostanoids) by these cells would bepredicted to yield benefit in rheumatoid arthritis.

Biological Assays

The compounds of this invention may be tested in one of severalbiological assays to determine the concentration of compound, which isrequired to have a given pharmacological effect.

NF-κB activity may also be measured in an electrophoretic mobility shiftassay (EMSA) to assess the presence of NF-κB protein in the nucleus. Thecells of interest are cultured to a density of 1×10⁶/mL. The cells areharvested by centrifugation, washed in PBS without Ca²⁺ and Mg²⁺ andresuspended in PBS with Ca²⁺ and Mg²⁺ at 1×10⁷ cells/mL. To examine theeffect of compound on the activation of NF-κB, the cell suspensions aretreated with various concentrations of drug or vehicle (DMSO, 0.1%) for30 min. at 37° C. prior to stimulation with TNF-α (5.0 ng/mL) for anadditional 15 min. Cellular and nuclear extracts are prepared follows.Briefly, at the end of the incubation period the cells (1×10⁷ cells) arewashed 2× in PBS without Ca²⁺ and Mg²⁺. The resulting cell pellets areresuspended in 20 uL of Buffer A (10 mM Hepes (pH 7.9), 10 mM KCl, 1.5mM MgCl₂, 0.5 mM dithiothreitol (DTT) and 0.1% NP-40) and incubated onice for 10 min. The nuclei are pelleted by microcentrifugation at 3500rpm for 10 min at 4° C. The resulting supernatant was collected as thecellular extract and the nuclear pellet was resuspended in 15 uL BufferC (20 mM Hepes (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl₂, 25% glycerol, 0.2 mMEDTA, 0.5 mM DTT, and 0.5 mM phenylmethylsulphonyl fluoride (PMSF)). Thesuspensions are mixed gently for 20 min at 4° C. then microcentrifugedat 14,000 rpm for 10 min at 4° C. The supernatant is collected anddiluted to 60 uL with Buffer D (20 mM Hepes (pH 7.9), 50 mM KCl, 20%glycerol, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF). All samples arestored at −80° C. until analyzed. The protein concentration of theextracts is determined according to the method of Bradford (Bradford,1976) with BioRad reagents.

The effect of compounds on transcription factor activation is assessedin an electrophoretic mobility shift assay (EMSA) using nuclear extractsfrom treated cells as described above. The double stranded NF-κBconsensus oligonucleotides (5′-AGTTGAGGGGACTTTCCCAGGC-3′) are labelledwith T₄ polynucleotide kinase and [g-³²P]ATP. The binding mixture (25uL) contains 10 mM Hepes-NaOH (pH 7.9), 4 mM Tris-HCl (pH 7.9), 60 mMKCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.3 mg/mL bovineserum albumin, and 1 ug poly(dI-dC)opoly(dI-dC). The binding mixtures(10 ug nuclear extract protein) are incubated for 20 min at roomtemperature with 0.5 ng of ³²P-labelled oligonucleotide (50,000-100,000cpm) in the presence or absence of unlabeled competitor after which themixture is loaded on a 4% polyacrylamide gel prepared in 1× Trisborate/EDTA and electrophoresed at 200 V for 2 h. Followingelectrophoresis the gels are dried and exposed to film for detection ofthe binding reaction.

The effect of compounds on the phosphorylation of IκB may be monitoredin a Western blot. Cellular extracts are subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels(BioRad, Hercules, Calif.) and the proteins transferred tonitrocellulose sheets (Hybond™-ECL, Amersham Corp., Arlington Heights,Ill.). Immunoblot assays are performed using a polyclonal rabbitantibody directed against IκBα or IκBβ followed with aperoxidase-conjugated donkey anti-rabbit secondary antibody (AmershamCorp., Arlington Heights, Ill.). Immunoreactive bands are detected usingthe Enchanced Chemiluminescence (ECL) assay system (Amersham Corp.,Arlington Heights, Ill.).

Assays for IκB kinases were-conducted as follows: IKK-α was expressed asa hexa-histidine tagged protein in baculovirus-infected insect cells andpurified over a Ni-NTA affinity column. Kinase activity was assayedusing 50 ng of purified protein in assay buffer (20 mM Hepes, pH 7.7, 2mM MgCl₂, 1 mM MnCl₂, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP,0.3 mM Na₃VO₄, 1 mM benzamidine, 2 μM PMSF, 10 μg/ml aprotinin, 1 ug/mLleupeptin, 1 ug/mL pepstatin, 1 mM DTT) containing variousconcentrations of compound or DMSO vehicle and ATP as indicated(Pharmacia Biotech Inc., Piscataway, N.J.). The reaction was started bythe addition of 200 ng IκB-GST (Santa Cruz Biotechnology, Inc., SantaCruz, Calif.), in a total volume of 50 uL. The reaction was allowed toproceed for 1 h. at 30° C. after which the reaction was terminated bythe addition of EDTA to a final concentration of 20 mM. Kinase activitywas determined by dissociation-enhanced lanthanide fluorescenceimmunoassay (Wallac Oy, Turku, Finland) using a phospho-IκB-α (Ser32)antibody (New England Biolabs, Inc., Beverly, Mass.) and anEu³⁺-labelled anti-rabbit IgG (Wallac Oy, Turku, Finland). The plateswere read in a VICTOR 1420 Multilabel Counter (Wallac), using a standardeuropium protocol (excitation 340 nm, emission 615 nm; fluorescencemeasured for 400 μs after a 400 usec delay). Data are expressed asfluorescence (cps) units.

IKK-β was expressed as a GST-tagged protein, and its activity wasassessed in a 96-well scintillation proximity assay (SPA). Briefly,IKK-β was diluted in assay buffer as described above (20 nM final), withvarious concentrations of compound or DMSO vehicle, 240 nM ATP and 200nCi [γ-³³P]-ATP (10 mCi/mL, 2000 Ci/mmol; NEN Life Science Products,Boston, Mass.). The reaction was started with the addition of abiotinylated peptide comprising amino acids 15-46 of IκB-α (AmericanPeptide) to a final concentration of 2.4 μM, in a total volume of 50 uL.The sample-incubated for one hour a 30° C., followed by the addition of150 uL of stop buffer (PBS w/o Ca²⁺, Mg²⁺, 0.1% Triton X-100 (v/v), 10mM EDTA) containing 0.2 mg streptavidin-coated SPA PVT beads (AmershamPharmacia Biotech, Piscataway, N.J.). The sample was mixed, incubatedfor 10 min. at room temperature, centrifuged (1000×g, 2 minutes), andmeasured on a Hewlett-Packard TopCount.

In addition, IKK-β or IKK-α activity is measured by phosphorylation ofrecombinant GST-IkappaBalpha using time-resolved fluorescence resonanceenergy transfer (TR-FRET) in 384-well microtitre plates. Briefly IKK-βorIKK-α is diluted in assay buffer (50 mM HEPES pH 7.4 containing 10 mMmagnesium chloride, 1 mM CHAPS, 1 mM DTT and 0.01% w/v BSA) to 5 nMfinal concentration. This is added to various concentrations of compoundor DMSO vehicle and the reaction started by addition of 25 nMGST-IkappaBalpha and 1 μM ATP in assay buffer to a volume of 30 uL.After incubation for 30 min at ambient temperature the reaction wasstopped by addition of 50 mM pH 7.4 EDTA (15 uL). Detection ofphophorylated product was achieved by addition of a LANCE europiumchelate labelled specific anti-phosphoserine monoclonal antibody at 0.5nM final concentration (Cell signalling Technology via Perkin Elmer) andallophycocyanin labelled anti-GST antibody at 10 nM final concentration(Prozyme) to give a final volume of 60 μl. After a further incubation atambient temperature of a least 30 min the signal was read on a PerkinElmer Discovery fluorimeter.

The effect of IKK-β inhibitors on primary synovial fibroblast mediatorproduction was assesses as follows: primary cultures of human RSF wereobtained by enzymatic digestion of synovium obtained from adult patientswith rheumatoid arthritis as previously described (Roshak et al.,1996b). Cells were cultured in Earl's Minimal Essential Medium (EMEM)which contained 10% fetal bovine serum (PBS), 100 units/ml penicillinand 100 μg/mL streptomycin (GIBCO, Grand Island, N.Y.), at 37° C. and 5%CO₂. Cultures were used at passages 4 through 9 in order to obtain amore uniform type B fibroblast population. For some studies, fibroblastswere plated at 5×10⁴ cells/mL in 16 mm (diameter) 24 well plates(Costar, Cambridge, Mass.). Cells (70-80% confluence) were exposed toIL-1β (1 ng/mL) (Genzyme, Cambridge, Mass.) for the designated time.Drugs in DMSO vehicle (1%) were added to the cell cultures 15 minutesprior to the addition of IL-1. Studies were conducted 3-4 times usingsynovial cells from different donors. RSF cellular extracts-wereprepared from cells treated as described above. Briefly, human RSF wereremoved by trypsin/EDTA, washed, and harvested by centrifugation.Cellular extracts were prepared as previously described (Dignam et al.,1983; Osborn, et al., 1989). Briefly, at the end of the incubationperiod the cells (1×10 ⁷ cells) were washed 2× in PBS without Ca²⁺ andMg²⁺. The resulting cell pellets were resuspended in 20 uL of Buffer A(10 mM Hepes (pH 7.9), 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM.

Effect of IKK-β inhibition on human monocyte stimulated eicosanoid andcytokine production was assessed as follows: monocytes were isolatedfrom heparinized whole blood by double gradient centrifugation aspreviously described. Isolated monocyte enriched PBMCs were then adheredto 24 well culture plates at 2×10⁶ cells/mL in RPMI 1640 10% FBS(Hyclone, Logan, Utah) for 2 h. to further enrich the monocytepopulation. The media was then removed, cells washed once with RPMI1640, and 1 mL RPMI 1640 10% FBS was added to the wells. Test compoundsare added to the wells with a final vehicle concentration of 0.05% DMSO.Monocytes were activated by the addition of 200 ng/mL endotoxin (LPS; E.coli serotype 026:B6)(Sigma, St. Louis, Mo.) and incubated for 24 hrs.Cell-free supernates were analyzed by ELISA for TNF-α (EIA developed atSB), PGE₂ (Cayman Chemical, Ann Arbor, Mich.), and IL-8 and IL-6Biosource International, Camarillo, Calif.). Viability of the cells wasdetermined by trypan blue exclusion.

Effect of IKK-β inhibitors on phorbol ester-induced inflammation wasassessed as follows: the inflammatory response induced by the cutaneousapplication of phorbol ester (PMA) to the external pinnae of Balb/c micehas proven to be a useful model to examine multifactorial inflammatorycell infiltration and inflammatory alteration of epidermis. The intenseinflammatory lesion is dominated by neutrophil infiltration, which canbe easily quantified by measurement tissue concentrationmyeloperoxidase, an azuriphilic granular enzyme present in neutrophils.In addition, the overall intensity of the inflammatory response can bemeasured by determination of ear thickness. Balb/c mice (n=6/group) wereadministered drug treatment or vehicle followed by PMA (4 ug/ear). Themice were sacrificed 4 h. later, the ear thickness determined and NF-κBactivation was monitored by IκBα western or EMSA analysis.

Effect of IKK-β inhibitors on rat carrageenan-induced paw edema wasassessed as follows: male Lewis rats (Charles River—Raleigh, N.C.) werehoused and allowed free access to food and water, and weighed between200-275 g for each experiment. Compound or vehicle (0.5% Tragacanth(p.o.) or 10% DMSO, 5% DMA, 30% Cremophor (i.p.)) was administered 30minutes to 1 hour prior to the carrageenan injection. Edema was inducedby injection of 1% carrageenan in sterile dH2O (0.05 ml/paw) into theplantar surface of the right hindpaw. Paw thickness was measured priorto administration of compound or vehicle, and again at 3 hours, todetermine change in paw volume. Rats were euthanized by CO2 inhalationand the right hindfoot was removed, immediately frozen in liquidnitrogen and stored at −80C for analysis.

To determine the effects of an IKK-2 inhibitor in the mousecollagen-induced arthritis (CIA) model, 12 male DBA/1 mice (20-22 grams)per treatment group were immunized on day 0 with a total of 100 uL ofcomplete Freund's adjuvant (CFA) containing 200 ug of bovine type IIcollagen. On day 21 mice were boosted with 100 uL of phosphate bufferedsaline (PBS) containing 200 ug of bovine type II collagen (the 100 uL ofcollagen/CFA or collagen/PBS was injected subcutaneously into the tail).The IKK-2 inhibitor in vehicle, or vehicle alone, was administeredintraperitoneally, twice daily, from days 1 through 40 (disease symptomsare evident beginning on days 25-28). Two additional treatment groupsincluded the positive control etanercept (Enbrel) (4 mg/kg,intraperitoneally, every other day), and the etanercept vehicle (PBS).Mice were scored daily, through day 50, for clinical symptoms (seebelow), and paw thicknesses were measured. In addition to the 12 miceper treatment group that were scored throughout the experiment, atseveral time points during the course of disease satellite mice (3-5 pertreatment group) treated as described above were utilized to measurecytokine/chemokine levels and p65 levels in the paw, the ex vivo antigenrecall response by draining lymph node antigen recall response bydraining lymph node.

Induction of arthritis: AIA is induced by a single injection of 0.75 mgof Mycobacterium butyricum (Difco, Detroit, Mich.) suspended in paraffinoil into the base of the tail of male Lewis rats aged 6-8 weeks (160-180g). Hindpaw volumes are measured by a water displacement method on day16 and/or day 20. Test compounds were homogenized in a suitable vehicleand administered by a suitable route. Control animals are administeredvehicles alone. Two dosing protocols are genrally used: prophylacticdosing, which is initiated on the day of adjuvant injection andtherapeutic administration, initiated on day 10 once inflammation hasbeen established.

Clinical Scoring

Each paw was assigned a score ranging from 0-4, based on the followingcriteria:

-   0=no inflammation-   1=single swollen digit-   2=several swollen digits, mild paw swelling-   3=several swollen digits, moderate paw swelling-   4=all digits swollen, severe paw swelling

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The above description fully discloses the invention including preferredembodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the followingclaims. Without further elaboration, it is believed that one skilled inthe area can, using the preceding description, utilize the presentinvention to its fullest extent. Therefore the Examples herein are to beconstrued as merely illustrative and not a limitation of the scope ofthe present invention in any way. The embodiments of the invention inwhich an exclusive property or privilege is claimed are defined asfollows.

1. A compound according to formula (I) hereinbelow:

wherein: R₁ represents NR₄R₅; R₂ represents CONH₂ or SO₂NH₂; R₃represents up to three substituents selected from the group consistingof halogen, C₁₋₄alkyl, NH₂, CF₃, OCF₃, O-alkyl, S-alkyl, CN, CHO,SO₂-alkyl, and NO₂; R₄ represents H, C₁₋₂ alkyl; R₅ representsC(=A)NHR₆, COR₇, or R₆; A represents O, S, or N; R₆ represents H, orC₁₋₂ alkyl; R₇ represents C₁₋₂ alkyl; L represents a linker D-E-D suchthat D represents a bond or C₁₋₄ alkyl; E represents

CONH, NHCO, COO, N, O, S, or

and G and I independently represent H, C₁₋₂ alkyl; or a pharamceuticallyacceptable salt thereof, provided that the compound of formula (I) isnot2-[(aminocarbonyl)amino]-5-{[(4-chlorophenyl)methyl]oxyl-3-thiophenecarboxamide.2. A compound according to claim 1 wherein R₂ is CONH₂.
 3. A compoundaccording to claim 1 wherein R₅ is C(=A)NHR₆.
 4. A compound according toclaim 1 wherein A is O.
 5. A compound according to claim 1 wherein E is

CONH, or,


6. A compound according to claim 1 wherein the compound is selected fromthe group consisting of:5-[(E)-phenyl)-ethenyl]-2-ureido-thiophene-3-carboxylic acid amide;5-[(E)-2-(4-Fluoro-phenyl)-ethenyl]-2-ureido-thiophene-3-carboxylicacidamide;5-[(E)-2-(4-Chloro-phenyl)-ethenyl]-2-ureido-thiophene-3-carboxylic acidamide; 5-Phenethyl-2-ureido-thiophene-3-carboxylic acid amide;5-Benzyl-2-ureido-thiophene-3-carboxylic acid amide;5-(1-Phenyl-ethyl)-2-ureido-thiophene-3-carboxylic acid amide;5-Phenylethynyl-2-ureido-thiophene-3-carboxylic acid amide;5-(4-Fluorophenylethynyl)-2-ureido-thiophene-3-carboxylic acid amide;5-(4-Ethylphenylethynyl)-2-ureido-thiophene-3-carboxylic acid amide;5-(4-Methoxyphenylethynyl)-2-ureido-thiophene-3-carboxylic acid amide;5-(4-Chlorophenylethynyl)-2-ureido-thiophene-3-carboxylic acid amide;5-(4-Trifluoromethylphenylethynyl)-2-ureido-thiophene-3-carboxylic acidamide;5-(3-Trifluoromethylphenylethynyl)-2-ureido-thiophene-3-carboxylic acidamide; and 5-Acetylamino-thiophene-2,4-dicarboxylic acid 4-amide2-[(3-chloro-phenyl)-amide]; or a pharmaceutically acceptable saltthereof.
 7. A method of treating a disease characterized by pathologicalNF-κB activation comprising inhibiting the pathological activation byadministering to a patient in need thereof an effective amount of acompound according to claim
 1. 8. A method according to claim 7 whereinthe disease is an inflammatory or tissue repair disorder.
 9. A methodaccording to claim 8 wherein the disease is selected from the groupconsisting of inflammatory and tissue repair disorders, particularlyrheumatoid arthritis, inflammatory bowel disease, asthma and COPD(chronic obstructive pulmonary disease) osteoarthritis, osteoporosis andfibrotic diseases, dermatosis, including psoriasis, atopic dermatitisand ultraviolet radiation (UV)-induced skin damage, autoimmune diseasesincluding systemic lupus eythematosus, multiple sclerosis, psoriaticarthritis, alkylosing spondylitis, tissue and organ rejection,Alzheimer's disease, stroke, atherosclerosis, restenosis, diabetes,glomerulonephritis, cancer, including Hodgkins disease, cachexia,inflammation associated with infection and certain viral infections,including aquired immune deficiency syndrome (AIDS), adult respiratorydistress syndrome, and Ataxia Telangiestasia.
 10. A method according toclaim 7 wherein said disease is COPD.
 11. A method according to claim 7wherein said disease is asthma.
 12. A method according to claim 7wherein said disease is rheumatoid arthritis.
 13. A method according toclaim 7 wherein said disease is dermatosis.
 14. A method according toclaim 7 wherein the disease is selected from the group consisting of:psoriasis, atopic dermatitis, and UV-induced skin damage.
 15. A methodaccording to claim 7 wherein the disease is selected from the groupconsisting of autoimmune diseases; tissue and organ rejection,Alzheimer's disease, stroke, atherosclerosis, restenosis, diabetes,glomerulonephritis, osteoarthritis, osteoporosis, and AtaxiaTelangiestasia.
 16. A method according to claim 7 wherein said diseaseis an autoimmune disease.
 17. A method according to claim 9 wherein theautoimmune disease is systemic lupus eythematosus, multiple sclerosis,psoriatic arthritis, or alkylosing spondylitis, diabetes
 18. A methodaccording to claim 7 wherein the disease is cancer and or cachexia. 19.A method according to claim 7 wherein the cancer is Hodgkins disease.20. A method according to claim 7 wherein the disease is inflammationassociated with infection and certain viral infections, includingacquired immune deficiency syndrome (AIDS).
 21. A method according toclaim 7 wherein the disease is AIDS.
 22. A method according to claim 7wherein the disease is adult respiratory distress syndrome.